Ischemic heart disease remains a leading cause of morbidity and mortality in the industrialized world, and prognosis after acute coronary syndromes is directly proportional to the extent of myocardial injury. A growing body of literature suggests that cardiac mitochondria are critical determinants of tissue viability. Recent clinical trials found that targeting mitochondria showed promise in reducing injury and improving patient outcomes. In spite of these exciting findings, the mechanisms that lead to mitochondrial dysfunction during the course of a myocardial infarction are not fully understood. In particular, there is a fundamental gap in our understanding of how changes in mitochondrial membranes directly hinder post-ischemic mitochondrial respiration. The long-term goal is to develop novel mitochondria-specific interventions that preserve cardiac tissue during times of metabolic stress. The objectives of this proposal are to elucidate the role of the mitochondrial membrane lipid environment on post-ischemic respiratory activity, and to determine if a mitochondria-directed peptide salvages tissue by optimizing lipid-dependent respiration. The central hypothesis is that post-ischemic mitochondrial respiratory function is compromised due to a disruption in the molecular organization of the inner mitochondrial membrane. This hypothesis is based on strong preliminary data showing ischemia-reperfusion decreases mitochondrial membrane fluidity, which prevents proper assembly of respiratory supercomplexes. Furthermore, preliminary evidence indicates that a cell- permeable, cardiolipin-targeted peptide protects the heart by rescuing the disruption in membrane fluidity. To accomplish the objectives, two specific aims will be tested. Specific Aim 1 will test the hypothesis that decreases in mitochondrial membrane fluidity promote mitochondrial dysfunction and reperfusion injury. Innovative approaches include assessment of mitochondrial membrane fluidity using both headgroup- and acyl side chain-sensitive probes, sophisticated imaging of cardiolipin dynamics in ventricular myocytes and intact hearts, and model membrane systems that recapitulate changes in heart mitochondria during ischemia-reperfusion. Specific Aim 2 will test the hypothesis that dysfunctional assembly of respiratory supercomplexes contributes to reperfusion injury. A comprehensive examination of post-ischemic respiration includes respiration studies in perfused hearts, permeabilized fibers, isolated mitochondria, and isolated respiratory supercomplex bands. The efficacy of cardiolipin-targeting peptide in preserving mitochondrial respiration will be tested vertically across models. The proposed research is significant as it is expected to expand understanding of the interaction of mitochondrial lipids and functional respirasomes during acute coronary syndromes. Ultimately, these studies have the potential to foster development of new therapies that reduce the burden of ischemic heart disease.